Although sickle cell disease and its clinical manifestations has been recognized within West Africa for several centuries, the first report of sickle cell anemia appearing in the medical literature occurred only in 1910 when James B. Herrick documented the presence of anemia in a 20-year old black male using photomicrographs illustrating the presence of "thin sickle-shaped and crescent-shaped" red cells (Arch. Intern. Med., 1910, 6: 517). Other cases of sickle cell disease were then continually recognized and reported over the next forty years until when in 1949 it was unequivocally confirmed that patients with sickle cell anemia had an electrophoretically abnormal hemoglobin, whereas those with the "sickle trait" had equal amounts of the normal and abnormal hemoglobin components. (Pauling, L., et al., Science, 1949, 110:543-548). The inheritance pattern of other hemoglobin variants was subsequently clarified and provided convincing evidence that hemoglobin (Hb) S and hemoglobin (Hb) C are allelic variants of normal hemoglobin.
Sickle cell anemia and the existence of sickle hemoglobin (Hb S) was the first genetic disease to be understood at the molecular level; and is recognized today as the morphological and clinical result of the glycine to valine substitution at the No. 6 position of the beta globin chain (Ingram, V M, Nature, 1956, 178:792-794). The origin of the amino acid change and of the disease state is the consequence of a single nucleotide substitution (Marotta et al, J. Biol. Chem., 1977, 252:5040-5053).
As sickle cell disease became better known and more easily identified, a remarkable degree of clinical heterogeneity in the physical manifestations and symptoms of sickle cell disease has become recognized. The anemia typically is of moderate severity and is usually well compensated by the dynamic steady state systems. The major source of mobidity and mortality is vaso-occlusion-which causes repeated episodes of pain in both acute and chronic form and also causes ongoing organ damage with the passage of time. Vascular occlusion often results in infarction of bone and/or bone marrow. Pulmonary and renal damage are frequently lethal in young adults; and cerebral infarction is often debilitating or fatal in children. Typically, patients afflicted with sickle cell disease are also very susceptible to bacterial infections and splenic dysfunction. Publications which describe the clinical and pathological manifestations in detail and review sickle cell disease are represented by the following: Clinton H. Joiner, Cation Transport And Volume Regulation In Sickle Red Blood Cells, American Journal of Physiology, 1992; Bunn, H F, and B. G. FoRget, Hemoglobin: Molecular, Genetic and Clinical Aspects, W. B. Saunders Co., Philadelphia, 1986, Chapters 11 and 12, pp. 453-564; Eaton, W A and Hofrichter, J., Blood, 1987, 70:1245-1266); and Hebbel, R P, Blood, 1991, 77:214-237); and the references cited within each of these publications.
It has long been recognized and accepted that the deformation and distortion of sickle cell erythrocytes upon complete deoxygenation is caused by polymerization and gelation of hemoglobin S. The phenomenon is well reviewed and discussed by Eaton and Hofrichter, Blood, 1987, 70:1245). To gain some perspective on the problem and consequences of Hb S polymerization and intracellular gelation, it is useful to consider the events believed to occur as a red cell travels through the circulation of a patient afflicted with sickle cell disease. Erythrocytes containing no polymerized hemoglobin S in the arterial circulation may pass through the microcirculation and return to the lungs without sickling; or they may sickle in the veins; or they may sickle in the capillaries. For purposes of description, sickling is equivalent with intracellular gelation. The probability for each of these possible events for the sickle red cell will be determined by the delay time for intracellular gelation relative to the appropriate capillary transit time (Eaton, W A, et al., Blood, 1976, 47:621). Thus, if it is thermo-dynamically impossible for intracellular gelation to take place, or if the delay time at venous oxygen pressures is longer than about 15 seconds, then cell sickling will not occur. Alternatively, if the delay time is between about 1 and 15 seconds, then the red cell will likely sickle in the veins. However, if the delay time is less than about 1 second, the red cell will sickle within the capillaries.
Note that for red cells that sickle within the capillaries, a number of possibilities exist as the consequent events-ranging from no effect on its transit time, to transient occlusion of the capillary, or to a more permanent blockage that may ultimately result in ischemia, or the infarction of the surrounding cells, and in destruction of the red cell. Which of these various possibilities and differing events will actually occur will depend on a number of factors: the total intracellular hemoglobin concentration; the composition of the intracellular hemoglobin; the rate and extent of deoxygenation; and the various transit times involved for the cells.
In addition, for unsickled red cells entering the microcirculation, a long capillary transit time will increase the probability of the potentially damaging vaso-occlusive events in two different ways. First, it will permit increased oxygen extraction which, in turn, will shorten the delay time. Second, it will increase the probability that a red cell with a given delay time will sickle within the capillary. Thus, for cells that either enter the microcirculation already sickled or become sickled within the microcirculation, there is a clear probability for occlusion of the small vessels; and the duration of an occlusion may be sufficiently long to comprise the oxygen supply to the surrounding tissues and hence alter the sickling a consequent vaso-occlusion in nearby microvessels. It is therefore critically important to recognize that vaso-occlusion is a dynamic process in which the fraction of capillaries that are occluded depends upon both rates of occlusion and the rate of capillary reopening. The factors that influence the transit times and the duration of occlusions thus play a critical role in the pathology in the sickle cell disease state.
It will also be noted and appreciated that the physical manifestations of sickle cell disease are paralleled by a cellular pathophysiology which is markedly diverse and varied. Certainly, much of the physiological dysfunction in sickle erythrocytes arises from the tendency of deoxy hemoglobin S to form an intracellular polymer-which results in a marked increase in cellular viscosity and impairment of rheological function. Sickle cells exhibit oxidative damage; abnormal adherence to endothelial cells, monocytes and other red cells; increased membrane rigidity; abnormal cytoskeleton function; deranged lipid structure; cation deletion and cellular dehydration; and abnormal carrier-mediated and passive permeability to cations.
Knowledge of the pathophysiology of sickle cell disease is merely one aspect of the continuing research interest in the physiology of erythrocyte cells generally. Considerable investigative efforts have focused upon the mechanisms of action and the various systems responsible for cation transport and volume regulation in normal red blood cells. In particular, the potassium transport pathways and the consequences of erythrocyte dehydration have been of major interest. A current summary of the various potassium transport pathways present in normal human erythrocytes is given by Table A below.
TABLE A Potassium Transport Pathways in Human Erythrocytes* Maximal Capacity as K+ transporter (mmol/ System Mode l.cells/h) Inhibitor Comments Reference NA+/K+ pump Normally 1-3 Cardiac ATP-driven: Glynn, L M, 3NA + 2K+ glycosides operates at The Enzymes but partial (ouabain) approx. 50% of Biological fluxes occur V.sub.max at normal Membranes. cell [Na+] 1985. NaKCl 1Na+:1K+:2C 0.1-1.5 Loop diuretics Poised at close Chipperfield, contransport 1-complex (bumetanide, to equilibrium AR, Clin. Sci. partial and furosemide) (i.e., zero net 71:465 (1986). exchange fluxes under fluxes physiological conditions) KCl 1K+:Cl- &gt;10 Internal Highest in Ellory, et al. cotransport divalent young cells; Biomed. cations; loop activated by Biochem. Acts diuretics at NEM, pressure, 46:531 (1987). high cell swelling, concentrations acid pH. Ca.sup.2 +-activated Uncoupled K+ &gt;10 Quinine Activated by Lew & Ferreira K+ channel raised cell Cur. Top. (Gardos [Ca.sup.2+ ] Memb. Transp. channel) 10:217 (1978). *Source: Stuart, J. and J. C. Ellory, Brit. J. Haematol. 69:1-4 (1988).
It has long been recognized that cytoplasm of the normal erythrocyte comprises approximately 70% water. Water crosses a normal erythrocyte membrane in milliseconds; however, the loss of cell water causes an exponential increase in cytoplasmic viscosity as the mean cell hemoglobin concentration (MCHC) rises above about 32 g/dl. Since cytoplasmic viscosity is a major determinate of erythrocyte deformability, the dehydration of the erythrocyte has substantial rheological consequences. Thus, the physiological mechanisms that maintain the water content of normal erythrocyte cells, and the pathological conditions that cause loss of water from erythrocyte cells in the blood circulation, are critically important. Moreover, since cell water will follow osmotically any change in the intracellular concentration of ions, the maintenance of the red cell's potassium concentration is of particular importance (Stuart, J., and Ellory, J C, Brit. J. Haematol., 1988, 69:1-4).
U.S. Pat. Nos. 5,273,992 and 5,441,957 describe the use of imidazoles, nitroimidazoles, triazoles and other aromatic compounds used in blocking of the Gardos channel, thus inhibiting the loss of K and preventing dehydration. The other major pathway responsible for the transport of K involves the K--Cl cotransporter system. The K--Cl cotransport system promotes loss of K and Cl with consequent erythrocyte dehydration when the cells are exposed to pH values lower than 7.4 (Brugnara, C., et al., J. Clin. Invest., 1985, 75: 1608-1617). To date, there exist no pharmacological inhibitors of this pathway that can be used to prevent cell dehydration. However, K--Cl cotransport is exquisitely sensitive to cell magnesium (Mg) concentration as well as other divalent cations, and a modest increase in cell Mg and/or other divalent cations induces marked inhibition of K--Cl cotransport in vitro (Brugnara, C., and Tosteson, D C, Blood., 1987, 70: 1810-1815). The Mg content of the red blood cell is an essential modulator of red blood cell volume, volume regulatory mechanisms, and enzymes involved in essential cellular metabolic functions. Erythrocyte Mg content also affects the activity of various membrane cation transport pathways such as the Na/K pump, Na--K--Cl cotransport, Ca and K channels, and K--Cl cotransport, and cell membrane structure and function. When Mg is increased, Cl moves into the cell to compensate the positively charged Mg ions with osmotically obligated water influx and consequent cell swelling.
A recent study using a transgenic mouse model for sickle cell disease (SAD 1 mouse, Trudel, M., et al., EMBO J., 1991, 11: 3157-3165), showed that a Mg-deficient diet led to worsening anemia, reticulocytosis, and increased dehydration of SAD 1 mouse red blood cells. By contrast, a high Mg-diet decreased K--Cl cotransport activity, red blood cell dehydration, and K loss of transgenic SAD 1 mouse red blood cells, and more importantly increased Hb levels, suggesting a possible amelioration of the disease (De Franceschi, L., et al., Blood, 1996, 88: 2738-2744).
In human patients, Mg supplements have been used to improve glucose oxidation in elderly, noninsulin-dependent (type II) diabetics (Paolisso, G., et al., J. Clin. Endocrinol. Metab., 1994,78: 1510-1514; Paolisso, G.,et al., Am. J Clin. Nutr., 1992,55: 1161-1167). Several uncontrolled studies that had focused on possible hemodynamic and vascular effects of various Mg preparations have been conflicting as to the benefits of Mg supplementation therapy in SS disease patients. Although initial reports indicated that orally or intravenously administered magnesium such as Mg citrate or Mg sulfate might be therapeutically beneficial for treating subjects with SS disease, subsequent studies have demonstrated that the therapeutic administration of Mg did not improve erythrocyte cell survival. (Anstall, H B, et al., Lancet, 1959, 1: 814-815; Lehmann, H., Br. Med J., 1963, 1: 1158-1159). For instance, a 7-day course of oral Mg supplementation using Mg citrate did not change erythrocyte survival in three patients with SS disease (Basu, A K, and Woodruff, A W, Trans. R. Soc. Trop. Med. Hyg., 1966, 60: 64-69). More recently, our own experiments showed no clinical benefits to sickle cell patients by the administration of Mg lactate (Brugnara, C., et al., unpublished observations).
Therefore, there exists a longstanding and well recognized need for an effective method of treating erythrocyte cell deformation in vivo, and in particular erythrocyte cells of subjects with a hemoglobinopathy such as sickle cell disease and thalassemia.